Optimized buffers and conditions to achieve high coverage of GC-rich regions

Build and receive within two weeks a panel for your unique content

The QIAseq Targeted DNA Panels have been developed as a complete Sample to Insight solution to enable digital DNA sequencing by utilizing molecular barcodes. Digital DNA sequencing is a unique approach to detect low-frequency variants with high confidence by overcoming the issues of PCR duplicates, false positives and library bias.

Each panel is a one-box, NGS platform-agnostic solution that contains all the necessary components to construct libraries from enriched genomic targets. Primer design is based on single primer extension, in which each genomic target is enriched by one target-specific primer and a universal primer, a strategy that removes conventional two target-specific primer design restriction and reduces the amount of required primers. All primers required for a panel are pooled into an individual primer pool to reduce panel handling and number of pools required for enrichment and library construction. Platform-specific indexes, which are contained in a separate box, allow the multiplexing of up to 384 samples per sequencing run. The unique buffer and enzyme system used in the QIAseq targeted DNA panels has been optimized to achieve high coverage of GC-rich genomic regions.

Custom panels can be designed to target exonic regions of genes, hotspots or SNPs, as well as intronic and promoter regions.

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Sensitivity: Digital DNA sequencing approach is optimized to deliver high confidence in calling low-frequency DNA variants. Over 90% sensitivity for 1% NA12878 SNP and indel on typical coding region with false positive less than 15 per mega base region when variants are detected with tiled primer design to cover complete coding region of each gene.

Universality: The chemistry used in the QIAseq Targeted DNA Panels and workflow is compatible with both regular and GC-rich genomic regions, allowing one to achieve 100% coverage of genes rich in GC content such as CEBPA and CCND1.

Flexibility: The QIAseq targeted DNA panels offer a high degree of flexibility in content and sample multiplexing. Several cataloged panels have been developed for a wide range of applications. One can also build a custom panel for a specific content, or extend the contents of an existing cataloged panel. Up to 384 samples can be multiplexed using the QIAseq indexes.

Principle

PCR duplicates are a major issue in targeted DNA sequencing, since, through PCR amplification, they turn unique DNA molecules into identical DNA molecules that cannot be distinguished from each other. In addition, errors from PCR amplification and sequencing process may also be present infinal reads that lead to false positive variants in sequencing results. This, in turn, results in the inability to confidently call DNA variants present at low frequencies in the starting DNA material. To overcome the issue of PCR duplicates and amplification artifacts, the QIAseq Targeted DNA Panels use digital sequencing by incorporating molecular barcodes into the starting DNA material before any amplification takes place, thereby preserving the uniqueness of the starting DNA molecules and overcoming the issues of PCR duplicates, false positives and library bias.

Procedure

The entire workflow of the QIAseq Targeted DNA Panels to go from extracted DNA to sequencing-ready libraries can be completed in 9 hours. Extracted DNA is fragmented, genomic targets are molecularly barcoded and enriched, and libraries are constructed. Sequencing files can be fed into the QIAseq pipeline, a cloud-based data analysis pipeline, which will filter, map and align reads, as well as count unique molecular barcodes associated with targeted genomic regions, and call variants with a barcode-aware algorithm. This data can then be fed into IVA or QCI for interpretation.

Applications

The QIAseq Targeted DNA Custom Panels can be used to call a variety of DNA variants from a wide range of sample types for numerous applications.